Preparative Columns 09 Overview of optimization methods for isolation/purification ----- 114, 115 YMC-Actus series ----------------------- 116~119 Ordering Information ------------------- 120~123 YMC-GPC series -------------------------- 124, 125 YMC-DispoPackAT ----------------------------- 126
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YMC GC Vol12 09 · 2016. 9. 12. · Preparative Columns 116 09 YMC-Actus series are semi-preparative HPLC columns that have excellent column durability and efficiency by applying
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Preparative Columns
09
Overview of optimization
methods for isolation/purification ----- 114, 115
YMC-Actus series ----------------------- 116~119
Ordering Information ------------------- 120~123
YMC-GPC series-------------------------- 124, 125
YMC-DispoPackAT----------------------------- 126
Preparative Columns
Preparative C
olumns
114
09
An overview of the methods for selecting optimum conditions for isolation/purification and conducting efficient isolation,
as well as points to check are given below.
Preparative packed columnsOverview of optimization methods for isolation/purification
Selection of a preparative column: General comments
1) Selecting the separation mode of chromatography
When more than one mode is available for the separation of samples, the following points should be considered for the selection of an
appropriate separation mode:
(1) Resolution : Selectivity of the packing material for the compound of interest
(2) Load : Capacity of the packing material
(3) Speed : Isolation time
2) Column size
The table shown in below provides a rough guide for selection of column inner diameters and packing material particle sizes.
(1) Column inner diameter : Sample load is proportional to the column cross-sectional area.
It is necessary to select a column inner diameter suitable for the sample load.
(2) Packing material particle size : Smaller particle gives higher column efficiency, however, costs higher and increases column
pressure. In addition, the equipment used needs to be resistant to the pressure.
When the target component and the nearest peak are very near and the highest resolution is needed,
packing materials with small particle size are useful.
In contrast, larger particle sizes result in lower column efficiency, but result in lower prices and lower
column pressure.
(3) Column length : Longer column gives higher resolution and higher sample load, but the column pressure becomes
higher and the separation takes longer.
The analytical conditions established using the analytical column are scaled up to the intended preparative scale in the direction shown by the arrow
Standard sample
load
Column efficiency Pressure
Cost
Most appropriate Appropriate Depending on purpose
5 10 10-20 15-30 50~Particle size
(µm)Inner diameter(mml.D.)
4.6 / 6.0
10 / 20
50
100-200
300-500
600~
tens of mg
hundreds of mg
g
hundreds of g
kg
up to tens of kg
General guidance for selection of preparative columns
Preparative C
olumns
115
Relationship between column inner diameter and flow rate/sample load
F : Analytical column flow rate (mL/min)F’ : Preparative column flow rate (mL/min)Dc : Analytical column inner diameter (mm)Dc’ : Preparative column inner diameter (mm)*Use the same equation to calculate the sample load.
When the same packing material and column length are used the preparative flow rate and sample load are proportional to the column cross-sectional area. Additionally, the resolution and column pressure experienced on the preparative column would be approximately the same as that experienced for the analytical scale separation.
Steps for performing a preparative separation
Consider screening packing materials that can be scaled for preparative isolations. If you suspect that larger quantities of the compound
needing purification will be required in the future, consider performing your analytical scale investigations on a packing material that
is available in preparative particle sizes (10 micron and larger). As the requirements for the purified product become greater at later
stages of the project, you will have the option to isolate larger quantities of material on larger particle sizes in larger columns on the
same packing chemistry. This is an important consideration if the compound should be required in much larger quantities as the project
matures.
Perform selectivity studies on analytical scale columns (4.6 mm I.D. and smaller) on a variety of packing materials under several sets of
conditions. Automated software programs may be particularly helpful in predicting the most desirable separation modes and in helping
to choose the best chromatographic conditions for your separations in the shortest amount of time.
Once the best resolution is obtained, perform loading studies on the analytical scale column. Evaluate product purity at variable loadings
and select the maximum load allowed for a desired product purity.
Select the size of preparative column that will be needed by scaling up the separation based on the loading obtained on the analytical
column. If possible, use the same particle size and column length to achieve predictable preparative results.
Scale up the loading of the preparative column based on the ratio of the cross-sectional areas of the preparative and anayltical columns.
Perform the preparative separation and evaluate the yield and purity.
1)
2)
3)
4)
5)
Preparative Columns
Preparative C
olumns
116
09
YMC-Actus series are semi-preparative HPLC columns that have excellent column durability and efficiency by applying axial
compression technology. YMC-Actus series columns show high durability under high flow rate or steep gradient conditions and
desirable for milligram scale preparative HPLC of various compounds.
High durability semi-preparative columns
Improved durability by applying axial compression technology
Prepacked column for milligram scale preparative HPLC
Excellent resolution
YMC-Actus seriesAxial Compression Technology for Ultimate Separation
Specification
Packing material Pore size(Å)
Particle size(μm)
C% UsablepH range Characteristics
Triart series
Triart C18 120 5 20
1.0~12.0
Superior peak shapeUsable over wide range of pH and temperatureUsable with 100% aqueous mobile phase
Triart C18 ExRS 80 5 25Excellent selectivity of isomers and structural analogsSuperior chemical durability
Triart C8 120 5 17Compete with the versatility of C18Usable over wide range of pH and temperatureIdeal for separations of isomers or structural analogs
Triart Phenyl 120 5 17 1.0~10.0Unique selectivity due to π-π interactionExcellent resolution without adsorption and tailing
Triart PFP 120 5 15 1.0~8.0Alternative selectivity to C18/C8 due to unique polar interactionSuperior planar cognitive ability / steric selectivityIdeal for separations of compounds or isomers
Pro series
Pro C18 120 5 162.0~8.0
High performance ODS packing material
Hydrosphere C18 120 5 12Can be used with 100% water mobile phaseSuperior separation for hydrophilic compounds
Pro C18 RS 80 5 22 1.0~10.0 High carbon ODS packing material, high durability
Pro C8 120 5 10 2.0~7.5Processed with advanced endcapping technologySuperior separation of basic compounds
YMC-Pack seriesODS-A 120 5 17
2.0~7.5Standard ODS from analytical to preparative
ODS-AQ 120 5 14 Good separation for hydrophilic compounds
Preparative C
olumns
117
Great durability achieved by applying axial compression technology
[Excellent durability provided by improved bed density]
Conventional Preparative Column
YMC-Actus
80
100
120
0
20
40
60
run
% o
f ini
tial t
heor
etic
al p
late
num
ber
Column durability study
0 200 400 600 800
Test condition
Sequential gradient test
x 100 run
Column performance test
Packing
YMC-Actus
Conventional Semi-preparative column
10% increase in bed density
Flow
No change
Degradation of performance
With compression
Withoutcompression
Attaching theend assembly
After continuous use under afast �ow rate at high pressure
Compression
initial
after 700 run
initial
after 300 run
Void/crack in packed bedFlow
Uniformly high density packing is necessary for high performance HPLC column. DAC (Dynamic Axial Compression) column is widely used for preparative separation in pilot or production scale. It allows uniformly high density packing and prevents formation of voids during use by applying continuous compression.YMC-Actus series have been developed by applying this Axial Compression Technology to semi-prep column. This column bed is compressed adequately by attaching the end assembly newly designed for YMC-Actus. It provides proper bed density (10% higher than conventional columns) and results in higher efficiency and durability.
Sequential gradient test(high-speed and high-pressure)Column size : 5 μm, 50 X 20 mmI.D. or 50 X 19 mmI.D.Eluent : A) water B) methanolGradient : 5%B (0-0.5 min),
Column performance testColumn size : 5 μm, 50 X 20 mmI.D. X or 50 X 19 mmI.D.Eluent : methanol/water (60/40)Flow rate : 10 mL/minSample : naphthalene
Preparative Columns
Preparative C
olumns
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09
6.7
pH 2.7
O
S
HN
Cl
CH3
OH
O
CH3
N
CH3
H3COH
OH
Clindamycin pKa=7.6
0.30
0.40
AU
0.00
0.10
0.20
0.30
0.40
min5.0 10.0 15.0
Clindamycin
2.00
2.50
3.00
AU
0.00
0.50
1.00
1.50
2.00
2.50
3.00
min5.0 10.0 15.0130222203522
130228101136
pH 9.8
pH
AU
0.00
0.10
0.20
0.30
0.40
AU
0.00
0.10
0.20
min5.0 10.0 15.0
AU
0.00
0.50
1.00
1.50
2.00
2.50
3.00
AU
0.00
0.50
1.00
1.50
min5.0 10.0 15.0
130227135854
130228181208
130227144916
min5.0 10.0 15.0 min5.0 10.0 15.0
0.0
2.0
4.0
6.0
8.0
(x 100)
13030633602
0.0 2.5 5.0 7.5 10.0 12.5 15.0 min
AU
0.00
0.10
0.20
0.30
0.40
130301211148
Purity : 100%Recovery : 86%
min0.0 4.0 8.0 12.0
Increasing loading amount
130306124728
Impact of loading amounton peak shape: High
Impact of loading amounton peak shape: Medium
Impact of loading amounton peak shape: Low
Effective for purificationat high loading
Purification at pH 9.8
Fraction analysis
Clindamycin
0.01 mg Loading (10 µL injection, 1.0 mg/mL in water) 0.5 mg Loading (10 µL injection, 50 mg/mL in water)
50 mg Loading(250 µL injection, 200 mg/mL in water)
YMC-Actus Triart C18 5 µm, 150 X 20 mmI.D.
Fraction
Separation at high loading
[Purification of basic pharmaceutical: Clindamycin]
Purification method developmentYMC-Triart C18 5 µm, 150 X 4.6 mmI.D.
Clindamycin and its impurities (related compounds) are more hydrophobic in their un-ionized form and are retained stronger at pH 9.8. At higher pH condition, the resolution between main peak and impurities is improved and the peak shape is less affected by increase of mass loading.Excellent chemical durability of YMC-Triart offers an option of purification at a high pH that is effective for basic compounds by increasing retention and mass loading. Moreover, highly efficient YMC-Actus Triart has identical performance to YMC-Triart analytical column. This enables direct scale up from analytical condition to preparative condition. The combination of YMC-Triart and YMC-Actus offers highly efficient purification of various compounds.
Eluent : A) 20 mM HCOOH for pH 2.7 20 mM HCOONH4 for pH 6.7 20 mM HCOONH4-NH3 for pH 9.8
B) acetonitrile 10-75%B (0-15 min)
Flow rate : 1.0 mL/min for 150 X 4.6 mmI.D. 18.9 mL/min for 150 X 20 mmI.D.
Temperature : 25°C for 150 X 4.6 mmI.D. ambient for 150 X 20 mmI.D.
Detection : UV at 210 nmPressure : 7.0 MPa for 150 X 4.6 mmI.D.
8.4 MPa for 150 X 20 mmI.D.
Column : YMC-Triart C18 5 μm 150 X 4.6 mmI.D.
Eluent : 50 mM KH2PO4 (pH 7.5 adjusted by 8 M KOH)/acetonitrile (55/45)
Purification of hydrophobic compounds with similar structure –Capsaicinoids in red pepper–Purification of hydrophobic compounds with similar structure –Capsaicinoids in red pepper–
Analysis
50 X 4.6 mmI.D. 2.0 mL/min, 20 µL injection
Brand G2 5 µm,YMC-Pack Pro C18 RS 5 μm50 X 4.6 mmI.D. 2.0 mL/min, 20 µL injection
Eluent : A) methanol/water/TFA (50/50/0.1) B) methanol/TFA (100/0.1) 10-30%B (0-5 min), 30%B (5-10 min)
Temperature : 25°C for 50 X 4.6 mmI.D. ambient for 50 X 20 mmI.D.
Detection : UV at 280 nmSample : methanol extract of a commercial cayenne pepper
(1 g cayenne pepper/3 mL)
Pro C18 RS has superior selectivity for hydrophobic compounds that differ slightly in structure and hydrophobicity, achieves better resolution between peak 1 and peak 2 .Furthermore, analytical separation can be directly scaled up to preparative scale with YMC-Actus Pro C18 RS. YMC-Actus series have high resolution equal to analytical columns.
Crude synthetic 30 mer oligonucleotide
5'-CCGCTCGAGCTAAAAAAAGCCTGTGTTACC-3'
Eluent : A) 10 mM DBAA* (pH 6.0)/methanol (60/40) B) 10 mM DBAA* (pH 6.0)/methanol (20/80) 10-35%B (0-30 min)
Temperature : ambientDetection : UV at 269 nmSample : synthetic oligonucleotide (100 μM)* di-n-butylammonium acetate
In analytical scale, many impurities could be separated from the target compound by onenucleotide difference on Hydrosphere C18. Even in purification scale, YMC-Actus gave superior separation and recovery.YMC-Actus Hydrosphere C18 is useful for purification of hydrophilic compounds such as oligonucleotides, organic acids, oligosaccharides and glycosides.
YMC-GPC series Suitable for separation of polymer or oligomer on the basis of molecular weight
Compatible with organic solvents with various polarities
High resolution and long lifetime under a high flow rate condition
High productivity by fast separation
Ideal for recycling GPC that can improve resolution
YMC-GPC is a column packed with highly cross-linked porous polystyrene/divinylbenzene media. It provides outstanding physical
rigidity for extended lifetimes especially at a high temperatures and in aggressive solvents. YMC-GPC offers high productivity on
preparative separation due to high resolution and high loadability, at a fast flow rate. Furthermore, higher resolution can be achieved
on a sample that is hardly separated in combination with recycling chromatography method, even without changing mobile phase
conditions or columns.
Polymer based Preparative GPC Columns
YMC-GPC has excellent solvent versatility. It can be transferred easily and rapidly between solvents of varying polarity. It is possible to select the optimum mobile phase depending on the solubility and separation behavior of the sample.
Calibration curves of polystyrene by YMC-GPC are shown left. The calibration curves are designed to be linear over a specified molecular weight range, ensuring that the same degree of resolution is achieved across the full operating range of the column. Desired separation can be achieved by selecting a column depending on the molecular weight range of a sample.
YMC-GPC is normally supplied in ethylbenzene unless otherwise stated. The transferring procedure to other mobile phases is described in the instruction manual on our website.
Recycling separation of polystyrene oligomers by YMC-GPC T4000 column is shown. By using recycling chromatography separation method, higher resolution can be achieved on a sample that is hardly separated, even without changing mobile phase conditions or columns. Furthermore, no solvent is consumed during recycling. It greatly contributes to reduction of solvent consumption on purification.
(※) See p.166, 167 for information of LC-Forte/R.
Recycling chromatography separation of polystyrene oligomers
0
100
200
300
400
500
mAU
0 20 40 60 min
1st
2nd
3rd4th
5th
G130904B
Ordering Information
YMC-GPC (Theoretical plate number > 20,000)
Product name Phase dimension
Molecular weight range (g/mol)
Column sizeinner diameter X length (mm) Product number
YMC-GPC T2000 50 Å~ 2,000
20 X 600 GP05S10-6020PTYMC-GPC T2000-40 10 μm 40 X 600 GP05S10-6040WTYMC-GPC T4000 100 Å
~ 4,00020 X 600 GP10S10-6020PT
YMC-GPC T4000-40 10 μm 40 X 600 GP10S10-6040WTYMC-GPC T30000 500 Å
500 ~ 30,00020 X 600 GP50S10-6020PT
YMC-GPC T30000-40 10 μm 40 X 600 GP50S10-6040WTYMC-GPC T60000 1000 Å
500 ~ 60,00020 X 600 GPA0S10-6020PT
YMC-GPC T60000-40 10 μm 40 X 600 GPA0S10-6040WTYMC-GPC T10M MIX
500 ~ 10,000,00020 X 600 GP9BS10-6020PT
YMC-GPC T10M-40 10 μm 40 X 600 GP9BS10-6040WT
Guard columns
Product name Particle size Column sizeinner diameter X length (mm) Product number
Guard columns YMC-GPC-G10 µm
7.5 X 50 GP99S10-05V5PTGGuard columns YMC-GPC-40G 25 X 25 GP99S10-G525PTG
Preparative Columns
Preparative C
olumns
126
09
Flash chromatography column
YMC-DispoPackAT Excellent resolution and reproducibility